High progesterone concentrations in serum throughout pregnancy are known to inhibit lactogenesis, yet progesterone levels remain elevated during lactation in several species. Evidence suggests that this regulatory effect is mediated via progesterone inhibition of glucocorticoid action. Detailed examination of progesterone competition for the glucocorticoid receptor in pregnant and lactating mammary glands has been hampered due to uncontrollable variables present in the in vivo system. These interactions can be studied within a controlled environment through the employment of a mammary epithelial cell line. The human MCF-7 cell line possesses both glucocorticoid and progesterone receptors and progesterone competes for the glucocorticoid receptor in this system. The experiments outlined in this proposal are designed to demonstrate that progesterone modulation of glucocorticoid-receptor interaction in human mammary epithelial cells represents the primary level of progesterone regulation of lactogenesis. The interaction between progesterone and glucocorticoids may be connected to a secondary level of control, e.g., modulation of prolactin-binding activity. The following model for progesterone regulation of glucocorticoid action will be examined: (1) modulation of cytoplasmic glucocorticoid-receptor interaction by progesterone through (a) alteration of receptor affinity for glucocorticoids, (b) regulation of glucocorticoid receptor levels, (c) depression of cytoplasmic progesterone receptor activity, thereby increasing the concentration of progesterone available to compete for the glucocorticoid receptor, and (d) fractional occupation of the glucocorticoid receptor by progesterone; (2) progesterone inhibition and/or competition for the glucocorticoid-receptor complex during activation, translocation, and nuclear binding; and (3) the mediation of progesterone effects through nuclear interaction of the progesterone receptor. These experiments will be correlated with the modulation of ultrastructural and biochemical features of secretion.